Prevention of muscular dystrophy in mice by CRISPR/Cas9–mediated editing of germline DNA

Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. Dystrophin is a large cytoskeletal structural protein essential formuscle cellmembrane integrity. Without it, muscles degenerate, causing weakness and myopathy.

DMD affects approximately 1 in 3500 boys, death of patients usually occurs by age 25, typically from breathing complications and cardiomyopathy. Hence, therapy for DMD necessitates sustained rescue of skeletal, respiratory, and cardiac muscle structure and function. Although the genetic cause of DMD was identified nearly three decades ago, and several gene- and cell-based therapies have been developed to deliver functional Dmd alleles or dystrophin-like protein to diseased muscle tissue, numerous therapeutic challenges have remained and presently and there is no effective treatment.

In a recent study, researchers used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)–mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germline of mdx mice, amodel for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene.The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle.With the anticipated technological advances that will facilitate genome editing of postnatal somatic cells, this strategymay one day allowcorrection of disease-causing mutations in the muscle tissue of patients with DMD. (Science, Sept 5 2014)

Sorting cells with sound waves

Acoustic device that separates tumor cells from blood cells could help assess cancer’s spread.

Researchers from MIT, Pennsylvania State University, and Carnegie Mellon University have devised a new way to separate cells by exposing them to sound waves as they flow through a tiny channel. Their device, about the size of a dime, could be used to detect the extremely rare tumor cells that circulate in cancer patients’ blood, helping doctors predict whether a tumor is going to spread.

Separating cells with sound offers a gentler alternative to existing cell-sorting technologies, which require tagging the cells with chemicals or exposing them to stronger mechanical forces that may damage them.

To sort cells using sound waves, scientists have previously built microfluidic devices with two acoustic transducers, which produce sound waves on either side of a microchannel. When the two waves meet, they combine to form a standing wave (a wave that remains in constant position). This wave produces a pressure node, or line of low pressure, running parallel to the direction of cell flow. Cells that encounter this node are pushed to the side of the channel; the distance of cell movement depends on their size and other properties such as compressibility.

However, these existing devices are inefficient: Because there is only one pressure node, cells can be pushed aside only short distances.

The new device overcomes that obstacle by tilting the sound waves so they run across the microchannel at an angle — meaning that each cell encounters several pressure nodes as it flows through the channel. Each time it encounters a node, the pressure guides the cell a little further off center, making it easier to capture cells of different sizes by the time they reach the end of the channel.


Want to live 100+ years!

By 2050, the number of people over the age of 80 will triple globally. These demographics could come at great cost to individuals and economies.

The problems of old age come as a package. More than 70% of people over 65 have two or more chronic conditions such as arthritis, diabetes, cancer, heart disease and stroke.

Restricting calorie intake in mice or introducing mutations in nutrient-sensing pathways can extend lifespans by as much as 50%. And these ‘Methuselah mice’ are more likely than controls to die without any apparent diseases.

The current tools for extending healthy life — better diets and regular exercise — are effective. But there is room for improvement, especially in personalizing treatments.

Longevity pathways identified in model organisms seem to be conserved in humans and can be manipulated in similar ways. Genetic surveys of centenarians implicate hormonal and metabolic systems. Long-term calorie restriction in humans induces drastic metabolic and molecular changes that resemble those of younger people, notably in inflammatory and nutrient-sensing pathways.

Several molecular pathways that increase longevity in animals are affected by approved and experimental drugs. Cancer and organ-rejection drugs such as rapamycin extend lifespan in mice and worms by muting the mTOR pathway, which regulates processes from protein synthesis to cell proliferation and survival. The sirtuin proteins, involved in a similar range of cellular processes, are activated by high concentrations of naturally occurring compounds (such as the resveratrol found in red wine) and extend lifespan in metabolically abnormal obese mice. A plethora of natural and synthetic molecules affect pathways that are shared by ageing, diabetes and metabolic syndrome. (Luigi Fontana, Brian K. Kennedy, Valter D. Longo, Douglas Seals& Simon Melov, Nature 511, 405–407 (24 July 2014) doi:10.1038/511405a)



Cell sugar coat and cancer

Cell membranes are covered with sugar-conjugated proteins. New findings suggest that the physical properties of this coating, which is more pronounced in cancer cells, regulate cell survival during tumour spread.

The cell membrane serves as a signalling interface that allows cells to exchange information with their environment. It is constructed from lipids and contains both transmembrane and lipid-tethered proteins, which can be further modified through the covalent addition of sugars to build glycoproteins. Cancer cells frequently have higher levels of glycoproteins, such as mucin-1, than do healthy cells, and individual glycoproteins can transduce environmental signals that directly promote malignancy. However, glycoproteins also collectively organize into a glycocalyx.

Integrins are transmembrane receptors that bind extracellular matrix (ECM) proteins and are key interpreters and integrators of both the biochemical composition and the mechanical properties of the extracellular space. Cells with a thick glycocalyx are more efficient at receiving cell-survival signals through integrins, owing to the kinetic-trap properties of the glycocalyx. This may facilitate metastatic spread by enabling cancer cells to survive in the varied tissue and fluid environments they must traverse to colonize distant organs.

Integrin-based cell-matrix signalling is important for many steps in metastasis, including the migration of cancer cells out of the primary tumour and through the ECM, their entry into the vasculature, survival in the circulation, adhesion to the vessel wall, exit from the vasculature, and migration to and proliferative expansion in a distant organ. By reducing the rate of integrin binding and promoting clustering at existing adhesion sites, bulky glycoproteins act to promote a stable interaction between the cancer cells and the ECM.

We expect that the optimal glycocalyx thickness for supporting different aspects of cancer-cell behaviour, including invasion, vascular spread and metastatic colonization, varies. But how cancer cells adapt their glycocalyx to the diverse surroundings that they experience during metastasis is an interesting open question. Andrew J. Ewald & Mikala Egeblad, Nature511,298–299(17 July 2014)doi:10.1038/nature13506)

Defects in in vitro generated stem cells

There are two methods for reprogramming mature cells to pluripotent stem cells, which can give rise to all cells of the body. The first direct comparison of the methods reveals that both can cause subtle molecular defects.

Pluripotent stem cells hold promise for disease modelling and therapeutics, because they have the potential to differentiate into almost all cell lineages. In particular, there is much interest in patient-derived pluripotent stem cells, which are genetically matched to the patient’s own cells, minimizing the risk of rejection by the immune system.

In the past decade, two cell-reprogramming methods have been successfully used to generate patient-derived pluripotent stem cells: (1) cloning and (2) direct reprogramming of differentiated cells to induced pluripotent stem cells, through the addition of a defined transcription-factor cocktail.

However, the molecular differences between cells derived using each method remain unclear.

Derivation of induced pluripotent stem (iPS) cells is an appealing technology, because iPS cells can be reproducibly derived from patient samples. But comparison of iPS cells with the pluripotent embryonic stem (ES) cells generated during normal embryogenesis shows that human iPS cells are not completely reprogrammed, and reveals epigenetic differences between the two cell types (epigenetic marks are lingering genomic modifications that affect gene expression without changing DNA sequence).

Cloning — also called somatic cell nuclear transfer (SCNT) — involves transfer of the nuclear material from a mature donor cell into an egg from which the nucleus has been removed. Pluripotent cells, called nuclear transfer ES (NT ES) cells, which are genetically matched to the donor, then arise as the egg begins to develop into an embryo. Generation of patient-specific NT ES cells from adult human cells is now feasible. Although SCNT does not involve introducing transcription factors that have the potential to cause cancer (which is a problem with iPS cell generation), the protocol is technically difficult.

Figure 1: Comparing techniques for generating stem cells.

Comparing techniques for generating stem cells.

Three techniques can be used to generate pluripotent stem cells in vitro:

a) Induced pluripotent stem (iPS) cells are generated from mature cells, which can be directly converted by the addition of a transcription-factor cocktail.

b) In somatic cell nuclear transfer (SCNT), the nucleus is removed from an egg and replaced with the nucleus from a mature donor cell. As this hybrid cell develops into an embryo, pluripotent stem cells called nuclear transfer embryonic stem (NT ES) cells can be extracted from a region called the inner cell mass (ICM).

c) Embryos derived from in vitro fertilization (IVF) give rise to IVF ES cells that can be extracted from the ICM.

Incomplete demethylation patterns correlated with abnormal gene transcription were observed in iPS cells. NT ES cells were more similar to IVF ES cells, although some transcriptional alterations were apparent in both reprogrammed cell types.

There is an abundance of factors that can be used to reprogram cells and expand them in vitro, and each can influence the epigenetic and functional properties of reprogrammed cells in distinct ways. This complexity disrupts simplistic attempts to define and obtain ‘perfect’ stem cells. (Vladislav KrupalnikJacob H. HannaNature, 511,160 – 162 (10 July 2014)) The Research article subject to this review: Ma et al., Abnormalities in human pluripotent cells due to reprogramming mechanisms, Nature, 511,177–183(10 July 2014)doi:10.1038/nature13551

Cancer’s madness

by Richard Saltus (HHMI Bulletin, Spring 2014)

Computational approaches reveal that massive chromosome alterations give cancer an edge.

Cancer cells are known for the rampant disorder in their genomes: extra or absent chromosomes or parts of chromosomes, long stretches of DNA gone missing or present in too many copies. “It looks like someone threw a stick of dynamite into the nucleus,” says HHMI Investigator Stephen Elledge of Harvard Medical School and Brigham and Women’s Hospital.

This chaotic state is called aneuploidy. It stems from errors during cell division causing the daughter cells to have abnormal numbers of chromosomes or chromosome fragments. Aneuploidy affects hundreds or thousands of genes and can wreak all kinds of havoc, including miscarriages, lethal birth defects, and disorders like Down syndrome.

Based on his group’s latest research, Elledge says these massive alterations have evolved because they give malignant cells an edge in the “brutal competition” to win out over normal cells.

Because chromosomes exist in pairs, the loss of single chromosomes affects only one copy of a given gene. The second copy on the partner chromosome remains intact. As a result, these “hemizygous” losses have a weaker effect on cancer growth than the mutation of both copies of a tumor suppressor gene. But the additive combination of groups of hemizygous losses can have a large impact.

We have basically answered the question: Does aneuploidy drive cancer? We believe it does” says Stephen Elledge.

To those familiar with the “two-hit” model of cancer, it may come as a surprise that loss of a single gene copy can have an effect. According to this model, a mutation in a single copy of a tumor suppressor gene does nothing because the second copy compensates, and only if that second copy is subsequently “hit,” or mutated, does the cell begin its malignant journey.

However, Elledge cites evidence that a large proportion of cancer-suppressing genes are “haploinsufficient”—loss of even one copy can contribute to cancer development. In fact, Elledge estimates that 30 percent of all genes in humans are haploinsufficient, which has important implications for human development and disease.

“Losing or gaining single copies of genes on their own may have small effects, but altering many at the same time gives the cancer cell an advantage,” says Angelika Amon, a biologist and HHMI investigator at the Massachusetts Institute of Technology who studies aneuploidy. “Once you see [Elledge’s findings], you realize these losses and gains are not random noise in tumors, and we can begin to understand them.” 

Precise division of a bacterial cell

The recent development of cell biology techniques for bacteria to allow visualization of fundamental processes in time and space, and their use in synchronous populations of cells, has resulted in a dramatic increase in our understanding of cell division and its regulation in these tiny cells. Cell division in bacteria is driven by a cytoskeletal ring structure, the Z ring, composed of polymers of the tubulin-like protein FtsZ, at the division site precisely at midcell. Z-ring formation must be tightly regulated to ensure faithful cell division, and several mechanisms that influence the positioning and timing of Z-ring assembly have been described. Several membrane-associated division proteins are then recruited to this ring to form a complex, the divisome, which causes invagination of the cell envelope layers to form a division septum.

Another important but as yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. How bacteria coordinate cell cycle processes with nutrient availability and growth is a fundamental yet unresolved question in microbiology. The deletion of the gene encoding pyruvate kinase (pyk), which produces pyruvate in the final reaction of glycolysis, rescues the assembly defect of a temperature-sensitive ftsZ mutant and has significant effects on Z-ring formation in wild-type B. subtilis cells. Addition of exogenous pyruvate restores normal division in the absence of the pyruvate kinase enzyme, implicating pyruvate as a key metabolite in the coordination of bacterial growth and division (mBio 2014).



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